Substrates containing polyphosphazene as matrices and substrates containing polyphosphazene with microstructured surface

ABSTRACT

The invention relates to substrates containing polyphosphazene with a forming surface as matrices for producing biological materials that can be implanted in a mammal. The invention also relates to a method for producing said substrates and substrates containing polyphosphazene with a microstructured surface.

This application was filed under 35 U.S.C. 371 as a national stage ofPCT/EP02/00230, filed Jan. 11, 2002; and which claims priority to GermanApplication No. 101 00 961.5, filed Jan. 11, 2001, respectively.

The present invention concerns substrates containing polyphosphazenewith a forming surface as matrices for producing biological materialsthat can be implanted in a mammal, a process for producing suchsubstrates, and substrates containing polyphosphazene withmicrostructured surfaces.

Culturing of cells, especially of endothelial cells, with the goal ofgrowing artificial organs, is a comparatively new development inimplantology. One particular advantage of this technology is thatimplants prepared in this manner are expected to exhibit completecompatibility with the body. Given that cell collections cultured exvivo initially do not have either the shape or the mechanical stabilitydesired for the later implants, such as organs, arteries, etc., suchimplants are initially preformed on a forming substrate. Examples ofsubstrates currently used, on which such cells are cultured, and whichare the primary supporting structure for such an implant, includepolylactides, polyethylene glycols, polyurethanes, Teflon, and inorganicsubstrates.

Numerous other materials being used to produce such primary supportingstructures or supporting substrates are known from the prior art and arebeing investigated. For example, an expandable shell of ε-PTFE, whichcan also be used for the culturing of artificial blood vessels, is knownfrom WO 98/56312. Other materials for this application are described inEP-A-0 810 845, U.S. Pat. No. 4,883,699, and U.S. Pat. No. 4,911,691.Examples of other polymers for this purpose include hydrolyzedpolyacrylonitrile (U.S. Pat. No. 4,480,642), hydrophilic polyethers(U.S. Pat. No. 4,798,876) and polyurethane diacrylates (U.S. Pat. No.4,424,395). Also, various hydrogels are known that can be used ascoatings for this purpose. The group of potentially applicable materialscan also be supplemented by polyvinylpyrrolidone (PVP), polyvinylalcohols (PVA), polyethylene oxide (PEO) and polyhydroxyethylmethacrylate p(HEMA). Furthermore, use of a group of standard materialssuch as polyurethanes, polyethylenes and polypropylenes is alsodescribed in the prior art as possible materials for such substrates.Mixtures of these materials with one another are also known. Anothergroup of materials is known from EP-A-0 804 909.

Given that the inherent characteristics of these materials differ, itcan be assumed that each of these materials, or each of thesesubstances, exhibits special characteristics for certain applications inthe culture of artificial implants. For instance, PVA dissolves verywell in liquid and is rapidly absorbed. Other materials exhibit goodcompatibility with blood. Still other materials are particularlyextensible. Unfortunately, however, all materials have drawbacks invarious areas. For instance, PVA does not exhibit particularly goodblood compatibility.

ε-PTFE, for instance, is quite extensible and also exhibits good bloodcompatibility; but this material is very difficult to handle andproduction of supporting substrates from this material requires a seriesof specific processing steps (see WO 96/00103). The surface of theε-PTFE substrate obtained in that manner is also very porous, so thatcells grow into this material very strongly and it is almost impossibleto avoid damage when separating the cultured cell material for animplant from the supporting substrate. For other materials, elasticproperties, which are important for such a supporting substrate in somecases, can be achieved only by adding plasticizers which reducecompatibility with the blood and body, and which also present anundesirable affect on the cell culture due to bleeding of the“plasticizers”.

The greatest difficulties that arise in the culturing of cells forimplants are reactions with the supporting substrate or with itsdegradation products. It is known, for example, that inflammatoryreactions can occur in recipients due to the dissolving or absorptionand decomposition of some of the substances known in the state of theart (van der Gieβen, Circulation, Volume 94, No. 7, 1996). Those ariseeither because of partially incomplete compatibility of such supportingsubstrates, or because of reaction with decomposition products thatarise due to decomposition of the substances noted. Furthermore, cracksand fractures can occur in the freshly cultured implant when thecultured implant is to be removed from the supporting substrate. Thatdisadvantageous effect is primarily due to the fact that the cellsgrowing for the implant bind very tightly to the supporting substrate,particularly so with polylactide, for example; because of the porestructure that arises from dissolution or due to the basic surfacenature of the supporting substrate, they intertwine with the supportingsubstrate so that it is practically impossible to remove them withoutdamage.

Behavior with respect to bacteria and proteins that are deposited on thesurfaces of the supporting substrate is also a major factor in thesuccessful culturing of the implants noted, cells in particular, becausethese deposits can lead to significant inflammations in patients and toother problems with the growth and culture of the cells.

The cracks that have been mentioned, which can occur on removal ofcultured blood vessel implants from the supporting substrate, are animportant aspect in the production of vascular implants. These cracksare, for instance, points of attachment for increased development ofthrombi in recipients or patients, and for other deposits (proteins,macrophages, etc.) that can become a risk for the recipients or patientsafter implantation.

Thus the present invention is based on the objective of providing a newsystem for producing implants from biological materials that is intendedto allow the most selective growth possible of the desired cells and toassure essentially damage-free separation of implants made of thedesired cells from the supporting substrate used.

This objective is achieved by the embodiments as characterized in theclaims. In particular, the use of a substrate with a forming (or formbuilding) surface is provided, comprising at least partially abiocompatible polymer with the following general formula (I)

in which n stands for 2 to ∞, R¹ to R⁶ are the same or different andstand for an alkoxy, alkylsulfonyl, dialkylamino or aryloxy group, or aheterocycloalkyl or heteroaryl group having nitrogen as the hetero atom,as a matrix for producing biological material that can be implanted in amammal.

In one preferred embodiment of the present invention, the biocompatiblepolymer according to Formula (I) is provided as a coating on thesubstrate to develop the forming surface. In this embodiment of theinvention, there is no particular limitation on the substrate used, andit can be any material, such as plastics, metals, metal alloys andceramics. The biocompatible coating has, for example, a thickness fromabout 1 nm up to about 1000 μm, preferably up to about 10 μm, andespecially preferably up to about 1 μm. In another preferred embodimentof the present invention the substrate having a forming surface is ashaped object or moulding or moulded article made of the biocompatiblematerial according to Formula (I).

The degree of polymerization of the biocompatible polymer according toFormula (I) is preferably in a range of 20 to 200,000, more preferablyfrom 40 to 100,000.

Preferably at least one of the groups R¹ to R⁶ in the polymer used is analkoxy group, substituted with at least one fluorine atom.

The alkyl groups in the alkoxy, alkylsulfonyl and dialkylamino groupsare, for example, straight-chain or branched-chain alkyl groups with 1to 20 carbon atoms, wherein the alkyl groups can, for example, besubstituted by at least one halogen atom, such as a fluorine atom.

Examples of alkoxy groups are methoxy, ethoxy, propoxy and butoxygroups, which can preferably be substituted by at least one fluorineatom. The 2,2,2-trifluoroethoxy group is particularly preferred.Examples of alkylsulfonyl groups include methylsulfonyl, ethylsulfonyl,propylsulfonyl and butylsulfonyl groups. Examples of dialkylamino groupsinclude dimethylamino, diethylamino, dipropylamino and dibutylaminogroups.

The aryl group in the aryloxy group is, for example, a compound with oneor more aromatic ring systems, in which the aryl group can, for example,be substituted with at least one alkyl group as defined above. Examplesof aryloxy groups are phenoxy and naphthoxy groups and derivativesthereof.

An example of the heterocycloalkyl group is a ring system containing 3to 7 atoms, with at least one ring atom being a nitrogen atom. Theheterocycloalkyl group can, for instance, be substituted with at leastone alkyl group as defined above. Examples of heterocycloalkyl groupsinclude piperidinyl, piperazinyl, pyrrolidinyl, and morpholinyl groupsand their derivatives. The heteroaryl group is, for example, a compoundwith one or more aromatic ring systems, in which at least one ring atomis a nitrogen atom. The heteroaryl group can, for example, besubstituted with at least one alkyl group as defined above. Examples ofheteroaryl groups include pyrrolyl, pyridinyl, pyridinolyl,isoquinolinyl and quinolinyl groups and their derivatives.

In another preferred embodiment of the invention, a layer containing anadhesion promoter is placed between the surface of the substrate and thebiocompatible coating made of the polyphosphazene derivative.

The adhesion promoter, or spacer, preferably contains a polar end group.Examples of those include hydroxy, carboxy, carboxyl, amino or nitrogroups. End groups of the O-ED type can also be used, in which O-EDmeans an alkoxy, alkylsulfonyl, dialkylamino or aryloxy group, or aheterocycloalkyl or heteroaryl group having nitrogen as a hetero atom,which can be variously substituted, for instance by halogen atoms,especially fluorine.

In particular, the adhesion promoter can, for example, be anorganosilicon compound, preferably an amino-terminated silane or onebased on aminosilane, amino-terminated alkenes, nitro-terminated alkenesand silanes or an alkylphosphonic acid. Aminopropyltrimethoxysilane isparticularly preferred.

The adhesion promoter in particular improves the adhesion of the coatingto the surface of the substrate by coupling of the adhesion promoter tothe surface of the substrate, through ionic and/or covalent bonds, forexample, and by further coupling of the adhesion promoter to thedescribed polymer of Formula (I) of the coating, for instance, throughionic and/or covalent bonds.

The term “biological material” includes, for example, eucaryotic cells,monolayer or multilayer cellular aggregations, tissues, or cellcomponents of mammals, especially of human origin. In one preferredembodiment the donor of the biological starting material is identical tothe recipient of the implantable biological material. Examples of thebiological starting material or biological material include endothelialcells of various origins (e.g., from skin, foreskin, blood vessels suchas the aorta, fatty tissues, eye, omentum, umbilical cord, varices, orthe like), epithelial cells of various origins (e.g., from the stomach,intestine, or the like), bone cells, cartilage cells, and all adherentcells or cells in which adherence is inducible, cell aggregations ortissues (e.g., artificial cultured skin or similar tissue), naturaltissues, proteins, sugar molecules and lipids. Artificial organs, bloodvessels, bones, cartilage, myelin sheaths, etc., can be produced byusing the substrate with a forming surface.

A further object of the present invention concerns a process forproducing the substrates with forming surface as defined above; whereinthe application of a coating of the biocompatible polymer according toFormula (I) to the surface of a shaping body, moulding (or mouldedarticle) or supporting substrate is known from the prior art.

For example, the substrate with a forming surface can be producedaccording to a preferred embodiment, in general, by the following steps:

-   (a) A solution containing at least one compound of the general    Formula (I) at a concentration of 0.1%-99% is prepared in a solvent    that is organic and polar. Ethyl acetate, acetone, THF, toluene, or    xylenes, for example, can be used here as solvents. Mixtures of    these solvents are also usable, or they can be supplemented by other    solvents. This solution is applied to a substrate that exhibits    little if any adhesion to the polymer, such as glass, silicon,    various ceramics or other appropriate materials such as polymers    (PDMS, Teflon, PMMA, polycarbonate or silicone). The surfaces of the    substrates listed can also be chemically modified, for instance, by    introducing certain functional groups (—NH₂, —OH, —COOH, —COH,    —COOMe, —CF₃, etc.).-   (b) Evaporation of the solvent can proceed without further measures;    but in the best case the concentration of the solvent vapor over the    substrate is controlled, as are the pressure and the temperature. At    the beginning of the first phase of drying, the atmosphere over the    coated substrate should be saturated with solvent vapor, with the    concentration of the solvent vapor then being reduced slowly over    many hours. The temperature can vary from −30° C. to +90° C. The    pressure can follow a gradient from normal pressure to water    aspirator vacuum (20 Torr) during the first phase of drying. After    the first phase of drying, the coated substrate is further dried for    a certain period at oil pump vacuum (0.1 Torr).

The substrate coated with the biocompatible polymer according to Formula(I) can then be used directly, without or after appropriatesterilization. Various coating thicknesses from 0.1 μm to 300 μm orthicker, preferably in the range from 0.5 μm to 30 μm, and especiallypreferably about 5 μm, are obtained, depending on the concentration ofthe polymer solution and the conditions used during the first phase ofdrying.

Another object of the present invention concerns a substrate with amicrostructured surface comprising at least partly a biocompatiblepolymer according to Formula (I) as defined above, with the size ormagnitude of the surface structures being in the range of nanometers,micrometers, or even larger or smaller, preferably in the range of 10 nmto 100 μM. In one preferred embodiment the biocompatible polymer ispresent on the substrate as a coating with an externally microstructuredsurface.

The structuring of the surface is not subject to any particularlimitation. For instance, all structures that can be generatedphotolithographically, with an electron beam, with an ion beam, with alaser, or by other techniques, can be produced. The microstructuring ofthe surface of the substrate or of the coating can also be obtained by“fusion structuring or melt structuring”, in which a thin wire isbrought to the melting temperature of the biocompatible polymer and thenmelts the desired structure into the surface of the coating by directcontact.

Special advantages can be attained by means of this structuring withstructures that affect the flow behavior of liquids particularlyfavorably (e.g., sharkskin or lotus effect) imprinted into the surfaceof the coating or substrate.

1. A method for producing an implantable biological material, the methodcomprising: providing a form-building surface of a substrate; disposinga polymer of general formula (I) to form a biocompatible coating layer,at least partially, above the form-building surface of the substrate;contacting donor cells of a biological starting material with thebiocompatible coating layer to promote the formation of cellularaggregations of the biological starting material above the biocompatiblecoating layer; and detaching in vitro the cellular aggregations of thebiological starting material from the biocompatible coating layer toproduce the implantable biological material, wherein the general formula(I) is

wherein n is 2 to ∞, and wherein R¹ to R⁶ are the same or different andrepresent a group selected from the group consisting of an alkoxy, analkylsulfonyl, a dialkylamino, an aryloxy, a heterocycloalkyl havingnitrogen as a heteroatom, and a heteroaryl having nitrogen as aheteroatom.
 2. The method of claim 1 further comprising disposing anadhesion promoter above the form-building surface and below thebiocompatible coating layer.
 3. The method of claim 2, wherein theadhesion promoter is aminopropyltrimethoxysilane.
 4. The method of claim1, wherein the contacting further comprises contacting the donor cellswith the biocompatible coating layer comprising the polymers of generalformula (I), wherein the biocompatible coating layer has amicrostructured surface.
 5. The method of claim 4, wherein thebiocompatible coating layer is micro-structured to exhibit surfacestructures of magnitude in the range of 10 nm to 100 μm.
 6. The methodof claim 1, wherein the biocompatible coating layer has a thickness from1 nm to 1000 μm.
 7. The method of claim 1, wherein the alkoxy group issubstituted by at least one fluorine atom.
 8. The method of claim 1,wherein the polymer is poly[bis(trifluoroethoxy)phosphazene].
 9. Themethod of claim 1, wherein the substrate is selected from the groupconsisting of glass, silicon, ceramics, polymers, metals, metal alloys,plastics, and combinations thereof.
 10. The method of claim 1, whereinthe substrate is chemically modified to introduce functional groups. 11.The method of claim 1, wherein the biological material is selected fromthe group consisting of eucaryotic cells, monolayer cellularaggregations, multilayer cellular aggregations, endothelial cells fromskin, endothelial cells from blood vessels, epithelial cells fromstomach, epithelial cells from intestine, bone cells, cartilage cells,myelin sheaths, and components thereof.
 12. The method of claim 1,wherein the biological implant is selected from the group consisting ofan artificial organ, an artificial blood vessel, an artificial bone, anartificial cartilage, and an artificial myelin sheath.